Fish-scale collagen peptides (FSCPs) were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromis sp.) scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the lighter FSCPs, 2000 and 1300 Da. In addition, the heavier seemed to preserve favorable coiled structures comparing to the lighter that presents mainly as linear under confocal scanning laser microscopy. FSCPs, particularly the heavier, were concluded to efficiently penetrate stratum corneum to epidermis and dermis, activate fibroblasts, and accelerate collagen synthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preserving the given desired structure feature.